Our laboratory studies center about two HTLV-I infected rabbit cell lines, one that mediates asymptomatic infection and another that causes acutely fatal disease or chronic cutaneous leukemia upon injection into rabbits. Molecular clones, K30p and K34p, were isolated from these HTLV-I infected rabbit cell lines and used to study HTLV-I infection in vitro and in vivo. Detailed functional comparisons are possible because clone K30p causes persistent infection in vitro and in vivo, whereas, clone K34p mediates only transient infection although the parent cell line is infectious. Comparison of the K30p and K34p nucleotide sequences reveal 18 base pair differences in LTR's, gag, pol and pX regions. Sequences of the 21 bp enhancer, tax, env and rex responsive element (RxRE) are identical in the two clones. In order to localize sequence substitutions controlling virus replication by the molecular clones, a series of chimeric HTLV-I clones containing various combinations of substitutions found in the two parent clones were constructed and characterized by transfection into a rabbit cell line RL-5. All transfectants produced intact virus particles in a transient fashion; only those with the entire protein coding region of K30p mediated virus integration, persistent virus production and reinfection. Clone K34p and all constructs with a single substitution (K227Q) in integrase (IN) failed to stably integrate suggesting that that this amino acid replacement in the integrase protein precludes provirus integration. Furthermore, clones containing either or both K34p specific base substitutions in a pX region sequence which encodes Rex and the regulatory proteins p13II and p30II failed to integrate and cause persistent virus production even though the clones encoded functional integrase. Infectious virus was produced by cotransfection using two defective clones with complementary IN and pX structures suggesting that their gene products could act in trans to mediate virus replication. Rabbits inoculated with cells transfected with two clones showed evidence for infection and new cell lines were derived from their PBMC. Provirus in these lines was a recombinant containing functional IN and pX genes. These data provide a clear rationale for the inability of the K34 clone to cause persistent infection in transfected cells and indicate minor replacements that preclude infection by HTLV-I molecular clones. Further studies will focus on exploring the host and viral factors that are involved in HTLV-I proviral integration. Studies are in progress to clone and express the integrase and the pertinent regulatory proteins of HTLV-I. These efforts will include use of methods to determine function of the these proteins.